Homo sapiens Raw sequence reads

The strict protospacer adjacent motif (PAM) requirement constrains CRISPR-Cas9 targeting. This study, presents a expand the PAM recognition range strategy by reconstructing PAM-interacting (PI) domain (REPAM) through ancestral sequence reconstruction technology. Using REPAM, the PAM specificity of Nme1Cas9 and Nme2Cas9 was simultaneously expanded from N4GATT/N4CC to N4CNH. Further mutation of the ancient PI domain of Nme2Cas9 expands the PAM to N4VHH. Subsequently, we applied it to SpaCas9, expanding its PAM recognition from NNGYRA to NNHH, increases theoretical target coverage by ~36-fold. Additionally, this SpaCas9 variant achieves editing efficiencies of up to 76.6% in HEK293T cells, substantially outperforming the SpRY variant. Moreover, base editors built based on SpaCas9 variant efficiently introduce protective genetic variants and precise splice-site mutations in cells. Overall, REPAM provides a generalizable and powerful platform for expanding the utility of CRISPR-based technologies.